Background: Recent studies have found deposition of phosphorylated α-synuclein (p-syn) in Parkinson disease (PD) patients' skin, indicating p-syn may be a potential biomarker of PD. However, the sensitivity of the p-syn detection varied largely from 5. 3 to 100%, this influenced the clinical use of this detection method to some extent.Objective: This study aimed to optimize the skin biopsy method for detecting p-syn deposition in patients with PD.Methods: Ninety PD patients and 30 healthy controls underwent skin biopsies at 2–3 of the following sites: the distal leg, thigh, cervical region, or forearm. Skin biopsy samples were cut to 50- and 15-μm thickness sections. Deposition of p-syn were detected by using double immunofluorescence labeling of protein gene production 9.5 (PGP9.5) /p-syn. Statistical data analysis was performed using SPSS 25.0 software.Results: Deposition of p-syn were found in 75/90 PD patients but not in healthy controls (p < 0.001). The positive deposition rate of p-syn in the single cervical site was significantly higher than that in the distal leg, thigh, and forearm site. Two samples from the cervical region had a higher p-syn positive rate compared to single cervical site . There was no significant difference between the p-syn positive rate of samples from the distal leg/cervical sites and 2 samples from cervical region . Next, the p-syn positive deposition rate of 2-biopsy samples including distal leg/cervical sites and double samples in the cervical site were comparable to the 3-biopsy samples. The 50-μm section had a significantly higher p-syn positive rate than the 15-μm section (p = 0.049).Conclusions: Two biopsy sites  or 2 samples from the cervical site were considered to be priority biopsy sites for detecting p-syn in PD patients. Thick sections may provide a higher p-syn positive rate than thin sections for skin biopsies. These findings provide an optimized p-syn detection method, indicate the valuable pathology biomarker of PD and will promote the clinical use of skin biopsy in the future. Parkinson disease (PD) is one of the most common neurodegenerative movement disorders . WorldwiThe main pathology characteristics of PD was the degeneration of dopaminergic neurons and the deposition of Lewy bodies in the substantia nigra. The main component of Lewy bodies is α-synuclein. Studies have reported that phosphorylated α-synuclein (p-syn) was identified by skin biopsy. The deposition morphology characteristics was identical to that of p-syn in the substantia nigra by autopsy , 6. P-syHowever, we found that the positive rate of p-syn deposits in the skin of PD patients varies from 5.3 to 100% due to the use of different biopsy sites, number of samples, and thickness of sections , 13, whiOur study aims to optimize the method for detecting p-syn in the skin of PD patients by comparing the p-syn positive rate of different biopsy sites, the number of samples biopsied, and the thickness of sections.PD patients irrespective of disease stage were continuously recruited from the in- or outpatient Department of Neurology in the First Affiliated Hospital of Zhengzhou University from February 2019 to February 2020 from skin biopsy database. Diagnosis of PD was based on the 2015 clinically established diagnostic criteria of the Movement Disorders Society  and was We collected general demographic information of PD patients. The motor symptoms of PD were assessed using the MDS Unified Parkinson's Disease Rating Scale III (MDS-UPDRS-III) . Hoehn &Three-millimeter punch biopsies were used to obtain the skin sites , 18. TheAll sections were analyzed and photographed under the Leica fluorescent microscope . The microscope settings were kept the same for all analyses. The deposition of p-syn was observed under high objective magnification (40 ×). The analysis was performed blindly by two examiners with expertise in immunofluorescence analysis, and the positive criteria reached complete agreement between the two analysts. The p-syn positive deposition ratio was expressed as a percentage of patients who had p-syn deposition in the skin nerves.p < 0.05 was used to define statistical significance.Statistical data analysis was performed using SPSS 25.0 software . Categorical data were tested by the χ2 test or Fisher's exact test. Numerical data were expressed as mean ± standard deviation. Non-parametric Mann–Whitney U tests were used to compute the continuous variables. A value of A total of 90 PD patients were included in this study, with a mean age of 59.81 ± 9.15 years . PD patients covered all H-Y stages with an average H-Y stage of 2.07 ± 0.81. Thirty healthy control subjects participated in the study. The demographic data are presented in p = 0.653), H-Y stage (p = 0.932), or the duration of the disease (p = 0.877) in PD patients between those with and without p-syn deposits.All PD patients received skin biopsies from the leg and cervical region. Among them, 24, 19, and 21 PD patients received an extra biopsy sample from thigh, forearm, and cervical area , respectively. There were no adverse events during or after the skin biopsies. There were no statistical correlations of the UPDRS score (p < 0.001). P-syn deposits were found in nerve bundles in both superficial and deep dermis, subepidermal plexus, and nerve fibers innervating blood vessels, sweat glands, arrector pili muscles and hair follicles, but not in intraepidermal nerve fibers. P-syn deposition in nerve fibers innervating sweat glands (A.b) and arrector pili muscles (B.b), and nerve bundles in deep dermis (C.b) was shown as representation in Dot-like or linear p-syn immunosignals colocalized with PGP9.5 signals in 75/90 (83.3%) PD patients, but in none of the controls (p = 0.001), the thigh , and the forearm site  among single-biopsy sites.The cervical site had a higher p-syn positive rate than the leg , and it was comparable with the cervical (single sample) or thigh site. Leg/cervical sites showed the highest p-syn positive rate in 2-site biopsies (1 sample in each site), which had significantly higher p-syn positive rate of the single leg (p = 0.001), thigh (p = 0.004), and the forearm (p = 0.003), respectively.The p-syn positive rate of the leg/forearm sites was significantly higher than that of the forearm site (p = 0.001), forearm/leg (p = 0.012), cervical/forearm (p < 0.001), and cervical/thigh (p = 0.021). Two samples taken from adjacent areas of the cervical site had higher p-syn positive rate compared with a single sample from this biopsy site . The p-syn positive rate was not different between 2-site leg/cervical biopsies and double samples from the cervical site  had significantly higher p-syn deposition than thigh/leg (p = 0.672) or thigh/leg  had higher positive rates than the 2-site biopsies. Combination of a third biopsy site of the forearm  or thigh  did not increase the p-syn positivity of the 2-site cervical/leg biopsy. Three samples, including two from the cervical region and one from the leg, did not further increase the p-syn positivity of the double samples from the cervical region  or 2-biopsy cervical/leg sites , but no differences were found in the leg or leg/cervical sites  from 16 PD patients were used to detect the p-syn positive deposition rate using section slices of 15- or 50-μm thickness. The 50-μm thick section had a higher p-syn positive rate than the 15 μm section in cervical biopsies  the highest p-syn positive rate was found in the cervical single-biopsy site; (2) 2-site leg/cervical biopsies and double samples from the cervical site had comparable p-syn positive rates, which were significantly higher than other 2-sample combinations in this study; (3) combination of a third biopsy site using the forearm or thigh did not increase the p-syn positivity of the 2-site cervical/leg biopsy; (4) the 50-μm thick sections had a higher p-syn positive rate than the 15 μm sections.Skin biopsies are minimally invasive, easy to perform, and allow multiple samples to be taken , 21. UsiThe results showed that the cervical site has the higher positive rate of p-syn than the thigh and distal leg which is consistent with the results of Donadio et al. and Mili et al. , 10. It Because small fiber neuropathy often occurred in PD patients, the leg was the recommended biopsy site for counting intraepidermal nerve fiber density and was considered the routine biopsy site . In our Zange et al.  found thThe thickness of most commonly used frozen sections were compared , 22, 23.Our study also existed some limitations. The participants recruited were all diagnosed according to clinical criteria without pathological confirmation by postmortem. However, this study provides systematic data on the skin biopsy method for detecting p-syn that supports the valuable potential of skin biopsy in early PD diagnosis, which may promote the clinical use of skin biopsies in the future.All datasets presented in this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by Ethics Committee of the First Affiliated Hospital of Zhengzhou University. The patients/participants provided their written informed consent to participate in this study.YX and JY contributed to conception, design of the study and manuscript revision. YX, JY, and YG contributed to supplement of the funding. XL, YY, QH, CJ, and LL organized the database, did the biopsies. XL, YY, and QH performed the experiments. XL, JY, YY, and YG performed the statistical analysis. XL wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It was confirmed that simulated microgravity (SMG) led to ultrastructural alterations and apoptosis in many types of microvascular endothelial cells. However, whether SMG would also affect choroidal vascular endothelial cells (CVECs) remains unknown. This study was designed to investigate the effects of SMG on ultrastructure and apoptosis of CVECs.The rotary cell culture system (RCCS) was utilized to simulate microgravity condition. Human CVECs were cultured under normal gravity (NG) or SMG condition for 3 days. The ultrastructure was viewed under transmission electron microscopy, and the organization of F-actin was observed by immunofluorescence staining. Additionally, the apoptosis percentage was calculated using flow cytometry. Moreover, the mRNA and protein expression of BAX, Bcl-2, Caspase3, Cytochrome C, p-AKT, and p-PI3K were detected with quantitative PCR and Western blot at different exposure time.P < 0.001). The mRNA and protein expression of Bax, Caspase3, and Cytochrome C of CVECs in SMG groups at 24 and 72 h significantly increased than those of the NG group, respectively (P < 0.001). The alterations of p-AKT and p-PI3K protein expression possessed similar trends. On the contrary, the mRNA and protein expression of Bcl-2 in CVECs under SMG at 24 and 72 h were significantly less than that of the NG group, respectively (P < 0.001).In the SMG group, CVECs presented with a shrunk cell body, chromatin condensation and margination, mitochondria vacuolization, and apoptotic bodies. The amount of F-actin decreased, and the filaments of F-actin were sparse or even partly discontinuous after cultivation under SMG for 72 h. The proportions of apoptotic CVECs in SMG groups at 24 and 72 h were significantly higher than those in the NG group (Simulated microgravity conditions can lead the alterations of the F-actin structure and apoptosis of CVECs. The Bcl-2 apoptosis pathway and PI3K/AKT pathway may participate in the damage of CVECs caused by SMG. It is well known that cardiovascular deconditioning occurs in astronauts exposed to microgravity. It might be ascribed to vascular endothelial cell (VEC) dysfunction as VECs orchestrate vessels and blood circulation, thus exerting a pivotal role on tissue homeostasis . VECs arThe cytoskeleton is known to be important for maintaining normal cell morphology and function. It was believed to play a vital role in adapting to external stress, including changes in gravity. It was reported that 22 s of weightlessness produced by parabolic flight maneuvers caused rearrangement of β-tubulin and altered morphology and gene expression of VECs . In humaSimulated microgravity conditions lead to not only alteration of cell morphology but also gene expression and then trigger apoptotic signals. It was demonstrated that SMG conditions upregulated the gene expression of FAS-L, p53, and BAX and downregulated the gene expression of PCNA and Bcl-2 in porcine aortic endothelial cells (PAECs) . SMG conTaken together, VECs may sense the altered circumstance forces brought by SMG and induce cytoskeletal alterations then activate some second messengers which proceed to result in various responses of genes and ultimately cause apoptosis.Choroidal vascular endothelial cells (CVECs), like the other kinds of VECs, make up the inner wall of choroidal vessels in the eyes and also play pivotal roles in regulating local blood flow as well as many physiological processes, which help to maintain tissue homeostasis. Particularly, its dysfunction may be associated with choroidal thickening which may exert influence on intraocular pressure and visual function which varied during and after spaceflight . However6 cells/vessel and cells attached to the microcarrier beads. The culture medium filled the culture chamber, and all air bubbles were removed. The rotation speed was set at 12 rpm/min. The control group sample  was placed in the same incubator. The rotation time lasted for 72 h.The RCCS bioreactor  was placed in a mixture incubator filled with CO2 and air. Microcarrier beads of 0.05 g Cytodex-3  were pretreated with 75% ethanol and washed with 0.1 mol/l PBS for three times before being added to the rotating culture vessel. Then, the CVECs were seeded to the culture vessel at a density of approximately 5 × 10The human CVEC line was obtained from Haling Biotechnology Co. Ltd (HL-CELL-0126) and was cultured in special basic medium and special additives  at 37°C; 10% fetal bovine serum (FBS), 100 mg/ml streptomycin, and 100 units/ml penicillin  were added to the medium.Choroidal vascular endothelial cells were washed with PBS and then fixed in 2.5% glutaraldehyde at 4°C for 24 h. After being dealt with 1% OsO4 for 1 h, CVECs were washed with PBS for 30 min and were dehydrated in graded ethanol series and then embedded in Epon-812. Sections (50 microns) were stained with 2% uranyl acetate solution and 1% solution of lead citrate for 30 min. At last, the ultrastructure was examined under transmission electron microscopy (TEM) .The F-actin of CVECs was observed by immunofluorescence staining. Briefly, CVECs were fixed with 4% paraformaldehyde for 30 min and then were incubated with a 5% FBS for 30 min at 37°C. Cells continued to be incubated with anti-F-actin  antibodies overnight at 4°C and incubated with FITC-conjugated anti-IgG  on the next day for approximately 1 h at 37°C and DAPI  for 2 min. Images were obtained using a confocal fluorescence microscope (Leica-LCS-SP8-STED).5 cells/ml. Following mixture with Annexin V-PE, the CVECs were incubated at 4°C without light for 10 min. Finally, CVECs were mixed with 7-AAD and were incubated for 5 min at 4°C in the dark. Flow cytometry was applied to detect the apoptotic cells.The CVECs were harvested using trypsin (0.25%) and washed in ice-cold PBS twice and suspended in binding buffer at a concentration of 5 × 10Total RNA of CVECs was extracted with TRIzol  and were reverse transcribed with the Prime Script RT Master Mix Kit  followed by the product instructions. The genes were then amplified from cDNA by PCR. The primers were as follows:Bcl-2: forward 5′-GGTGGGGTCATGTGTGTGG-3′ and reverse 5′-CGGTTCAGGTACTCAGTCATCC-3′Bax: forward 5′-CCCGAGAGGTCTTTTTCCGAG-3′ and reverse 5′-CCAGCCCATGATGGTTCTGAT-3′Cyto-c: forward 5′-ACCAGGCTCACATGCCCTA-3′ and reverse 5′-TTCGATGTCACGGGATGTCAT-3′Caspase3: forward 5′-CATGGAAGCGAATCAATGGACT-3′ and reverse 5′-CTGTACCAGACCGAGATGTCA-3′GAPDH: forward 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′.® Prime Script® PLUS RT-RNA PCR Kit . GAPDH was used as the internal control, and the relative expression of mRNA was calculated using the 2–ΔΔCT method.The steps were as follows: after denaturation (5 min at 94°C), amplification , and extension (72°CC 5 min); RT-qPCR was conducted with the One Step SYBRThe CVEC proteins were lysed in RIPA buffer containing protease inhibitors. Then, the protein concentrations were detected using the BCA Protein Assay Kit . Next, the protein sample was electrophoresed by 8% SDS-PAGE and blocked with 5% bovine serum albumin and then was incubated with the primary antibody: anti-Bcl2 , anti-Bax , anti-Caspase 3 , anti-Cytochrome C , anti-p-AKT , anti-p-PI3K , and anti-GAPDH . Then, the primary antibody was washed out for 3 times and the membranes were incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody  for 1.5 h at room temperature. The intensity of immune response was observed the with Image J software .t-test was used to determine significance of difference. All experiments were performed three times, and P < 0.05 was considered as a statistical difference.The data were expressed as the mean ± SD and were analyzed using SPSS 11.5 software . One-way analysis of variance followed by Student’s Transmission electron microscopy was employed to observe the ultrastructure of CVECs. The cells in the NG group were characterized with complete nuclear membrane, abundant mitochondria, and much endoplasmic reticulum . After bWe performed immunofluorescence staining to determine whether there were alterations of F-actin, one of the major cell cytoskeletons, in CVECs under SMG. It showed that CVECs in the NG group exhibited high fluorescence intensity and intact fiber appearance . In contP < 0.001). Moreover, in the SMG group the proportion of apoptosis at 72 h was significantly higher than that at 24 h (P < 0.001)  .P < 0.001). Moreover, in SMG groups, the relative mRNA expression levels of these genes of CVECs at 72 h were significantly higher than those at 24 h (P < 0.001). The relative Bcl-2 mRNA expressions of CVECs in SMG groups at 24 and 72 h were significantly lower compared with those in the NG group, respectively (P < 0.001). Moreover, the relative Bcl-2 mRNA expression of CVECs at 72 h in the SMG groups was significantly lower than that at 24 h (P < 0.001)  .P < 0.001). Moreover, in the SMG groups the protein expression at 72 h was significantly higher than that of 24 h (P < 0.001). In contrast, the relative Bcl-2 protein expression of CVECs in the SMG groups at 24 and 72 h were significantly lower than those in the NG group, respectively (P < 0.001). Moreover, in the SMG groups, Bcl-2 protein at 72 h was lower than that at 24 h (P < 0.001)  .The study demonstrated that SMG caused ultrastructural changes in CVECs including cell shrinkage, chromatin condensation and margination, mitochondrial cavitation, and apoptotic bodies. Besides, the proportion of apoptotic CVECs under SMG increased. These observations suggested that CVECs are sensitive to SMG and SMG conditions promote CVEC apoptosis. These findings were in accordance with previous researches in many other kinds of cells under SMG . HoweverHowever, the mechanism of cell apoptosis induced by SMG is unclear. Some pro-apoptosis genes and miRNA have been found upregulated in cells under SMG which can partly elucidate the enhanced apoptosis . For exaIn this study, the filaments of F-actin in SMG-exposed CVECs were found sparse and even partly discontinuous as a sign of F-actin depolymerization, demonstrating structural alteration of F-actin. Additionally, the decreased amount of F-actin was confirmed with fluorescence intensity analysis. These findings suggest that F-actin cytoskeleton alteration associated with CVEC apoptosis was induced by SMG. However the detailed mechanism remains unclear.It is well known that harmful stress induces apoptosis in cells via the mitochondria pathway and/or Bax pathway. Bax triggers cytochrome C release from mitochondria. In contrast, Bcl-2 inhibits cytochrome C release through stabilizing the mitochondria membrane. Then, the initiator caspase is activated by the released cytochrome C. Soon after that, the effector caspase such as caspase 3 activates Dnase, ultimately resulting in DNA fragmentation. Thus Bax, cytochrome C and caspase 3 exert pro-apoptotic and Bcl-2 exerts anti-apoptotic effects in the mitochondrial pathway .In the present study, we further examined the mitochondria pathway. It showed upregulation of Bax, cytochrome C, and caspase 3 in both gene and protein expression in CVECs under SMG. The results are consistent with the previous studies in other cell types. Many kinds of cells have demonstrated similar alterations including human osteoblastic cells , HUVECs We further examined the activated form of PI3K and Akt. The PI3K/Akt pathway regulates cell survival, proliferation, and motility. The PI3K-Akt pathway has been indicated in participating in eNOs production and inhibition of apoptosis in SMG exposed-cells . In the in vivo. Microgravity is not the only change of circumstance for the cells. Microgravity fluid shift was also believed to exert significant changes in eyes during and after long-duration spaceflight (in vivo and long-term studies under stimulated microgravity and in real spaceflight should be carried out.It is well accepted that the endothelium as a barrier lining the inner side of blood vessels orchestrates vessels and blood circulation, thus maintaining tissue homeostasis. Cell apoptosis and cytoskeleton disorganization as well as reduction would cause compromised function of choroidal endothelial cells and then might increase permeability and disrupt the integrity of vessel walls. Besides, as the cytoskeleton changes have direct influence on signal transduction, cytokine synthesis and secretion, and activated PI3K/Akt pathway regulating many cell activities, SMG appears to have potential for dysfunction of CVECs, choroidal vessels, and choroid. However, this association needs more evidence. As we know, it would be more complicated ceflight . TherefoTaken together, our results show identified increased apoptosis; reduction and disorganization of F-actin filament; downregulation of Bcl-2 gene expression; upregulation of BAX, Caspase3, and Cytochrome C gene expression; increase in BAX, Caspase3, Cytochrome C, p-AKT, and p-PI3K protein level; and downregulation of Bcl-2 protein level in CVECs under SMG. It indicates that during the process the Bcl-2 apoptosis pathway is triggered and the PI3K/AKT pathway is concomitantly upregulated probably to counter apoptosis in this process. This study is the first to investigate the role of SMG provided by RCCS on CVECs. These altered genes and proteins reported here might provide some new insights into the mechanisms underlying microgravity-induced changes in eyes and offer opportunities to develop countermeasures.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.in vitro cell experiments. All authors have read and approved the content of the manuscript.LL was the corresponding author, carried out the concepts, design, definition of intellectual content, and reviewed manuscript. HZ and YS were co-first authors, and they both participated in the design of this study, performed the data acquisition, statistical analysis, and manuscript preparation. CQ and JZ performed statistical analysis and drafted the first manuscript. YG and CN carried out the animal experiments and collected important background information. BW, YY, and FW conducted all of the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background and Aims: Lactose intolerance (LI) is a global problem affecting more than half of the world’s population. An ultra-purified, high-concentration galacto-oligosaccharide, RP-G28, is being developed as a treatment for patients with LI. The efficacy and safety of RP-G28 in reducing symptoms of lactose intolerance were assessed in a blinded, randomized, placebo-controlled trial. Methods: In this multiclinical site, double-blinded, placebo-controlled trial, 377 patients with LI were randomized to one of two doses of orally administered RP-G28 or placebo for 30 days. A LI test and symptom assessment were performed at baseline and on day 31. The primary endpoint was a ≥4-point reduction or a score of zero on LI composite score on day 31. Voluntary milk and dairy intake and global outcome measures assessed patients’ overall treatment satisfaction and quality of life before therapy and 30 days after therapy. This study received Institutional Review Board (IRB) approval. Results: For the primary endpoint, 40% in the RP-G28 groups reported a ≥4-point reduction or no symptoms on LI symptom composite score compared to 26% with placebo (p = 0.016). Treatment with RP-G28 also led to significantly higher levels of milk and dairy intake and significant improvements in global assessments compared to placebo. RP-G28 but not placebo led to significant increases in five Bifidobacterium taxa. Conclusions: RP-G28 for 30 days significantly reduced symptoms and altered the fecal microbiome in patients with LI. Treatment with RP-G28 also improved milk/dairy consumption and quality of life and was safe and well tolerated. Non-persistence of the lactase enzyme in the small intestinal mucosa (LNP) affects 65–70% percent of the population globally, impacting health and potentially causing distressing gastrointestinal (GI) symptoms, which are commonly referred to as lactose intolerance (LI) . LNP resPatients with LI often avoid dairy products. Prolonged restriction of dairy can result in insufficient dietary calcium and vitamin D, which can, in turn, lead to osteoporosis, osteomalacia, and hypertension ,4. The dLactobacillus and Bifidobacterium enhance β-galactosidase activity and GOS utilization [There is accumulating evidence that treatments which alter the gut microbiome can improve a wide range of diseases, either by reversing disease-associated alterations in the microbiome or “dysbiosis” or by modifying the “normal” gut microbiome. One example of this latter strategy includes galacto-oligosaccharides (GOSs), which pass intact through to the colon, where they stimulate the growth of lactose-metabolizing bacteria. Elevated populations of lization ,8, resullization .p = 0.019). Additionally, RP-G28-treated patients were 6 times less likely than patients given placebo to report LI 30 days post-treatment after reintroduction of dairy foods (p = 0.039).RP-G28, an ultra-purified, high-concentration (>95%) galacto-oligosaccharide, has been evaluated in a Phase 2a study in 85 patients [The aim of the current study was to further evaluate the efficacy and safety of two doses of RP-G28 in patients with LI in a larger, multicenter, randomized, double-blind, placebo-controlled, parallel group trial.A multicenter, randomized, double-blind, placebo-controlled, parallel group clinical trial was conducted to determine the efficacy, safety, and tolerability of two doses of RP-G28 in subjects with moderate to severe LI. The study took place between March and October 2016 at 15 investigative centers throughout the U.S. and included a 7 day screening, a 30 day treatment, and a 30 day post-treatment “real-world” observation, during which dairy was re-introduced to patients’ diets .The clinical trial consisted of 3 phases: a screening phase, a treatment phase and a post-treatment phase. Patients were screened for LI for 7 days prior to study. Patients were then stratified into higher and lower-dose RP-G28 treatment or placebo for a 30 day treatment phase, during which patients did not consume lactose. On day 31, post-treatment, LI symptom assessments were made. Following this, “real-world” dairy intake began, and LI symptoms were assessed over a 30 day period. www.clinicaltrials.gov), trial number NCT02673749.Study patients, investigators, study site staff, the sponsor, the medical monitor and the study monitors were blinded to the treatment during the trial. All sites obtained Institutional Review Board (IRB) approval. This investigation was carried out in accordance with the Declaration of Helsinki of the World Medical Assembly and its revisions, as well as the rules of Good Clinical Practice (GCP) of the United States FDA . IRB approval for protocol G28-003 was approved on 26 January 2016, with approval of Amendment #1 on 5 May 2016, and Amendment #2 on 17 June 2016. This clinical trial can be found on the clinical trial registry website  following a standardized in-clinic lactose challenge. The criteria for diagnosis of LI, and the main criteria for inclusion into the study, are provided in A 5 h HBT at baseline confirmed LNP in patients. Hydrogen, methane, and carbon dioxide concentrations were measured in exhaled breath following a single-blinded lactose challenge three times during the study. Anhydrous food-grade lactose was administered at 0.35/kilogram body weight on day 0 , on day 31 , and on day 61 (end of post-treatment period). After fasting overnight, patients were assessed for symptoms of LI using a validated symptom questionnaire (LI Symptom Questionnaire) to create a composite symptom score  at 6 time points starting at 30 minutes and hourly thereafter for 5 h.Subjects who met the eligibility criteria completed a global assessment questionnaire and a 7 day lactose consumption assessment recall, provided stool samples, and were randomized to 1 of 2 dose regimens (higher or lower) of RP-G28 or placebo in a 1:1:1 ratio via a centralized, randomized Interactive Response (IXR) system using the subject ID number assigned at the beginning of screening. The IXR system was then used to assign drug kit numbers.Subjects were asked to refrain from ingesting lactose-containing beverages/foods during the treatment period and record LI symptoms and adverse events (AEs) on a daily basis. The first dose of the study drug was administered on day 1 and the final dose on day 30. Subjects were followed for an additional 30 days after the final dose, during which time subjects were encouraged to ingest lactose-containing foods and record symptoms of LI daily. The demographics and characteristics of the study population are provided in The lower dose of RP-G28 was 5 grams twice daily on days 1–10 followed by 7.5 grams twice daily on days 11–30. The higher dose of RP-G28 was 7.5 g twice daily for days 1–10, followed by 10 g twice daily on days 11–30. The placebo  was administered in a blinded matching packet.The amount of lactose consumed each day for 7 days pre-treatment, and for 30 days post-treatment, was assessed via a food diary. Further, during the 30 day post-treatment period, global patient assessment questionnaires measured feelings, experiences and dietary changes resulting from treatment . Global 10 Each 16S rRNA gene amplicon was purified using the AMPure XP reagent . Next, each sample was amplified using a limited cycle Polymerase Chain Reaction (PCR) program, adding Illumina sequencing adapters and dual-index barcodes (index 1(i7) and index 2(i5)) (Illumina) to the amplicon target. The final libraries were again purified using the AMPure XP reagent, quantified and normalized prior to pooling. The DNA library pool was then denatured with NaOH, diluted with hybridization buffer and heat denatured before loading on the MiSeq reagent cartridge and on the MiSeq instrument (Illumina). Automated cluster generation and paired-end sequencing with dual reads were performed according to the manufacturer’s instructions.The 16S rRNA amplicon sequencing was performed utilizing patient stool samples collected on days 0, 31 and 61. Amplification using universal primers targeting the V4 region of the bacterial 16S rRNA gene was performed on 12.5 nanograms of total DNA from collected samples. Bifidobacteria and Lactobacilli using specific 16S rRNA gene and GroEL probes [The 1050 DNA samples corresponding to 345 subjects receiving placebo, lower-dose or higher-dose treatments at three time points  were analyzed by high-throughput quantitative PCR (qPCR) targeting L probes ,12. MicrA LI Symptom Questionnaire was developed, validated and applied during this clinical trial. This questionnaire rated individual symptoms of LI on an 11-point Numerical Response Scale (NRS) as well as a Verbal Rating Scale (VRS).The primary efficacy endpoint was the proportion of responders on day 31. A responder was defined as a patient with a reduction from baseline in the composite score of 4 points or greater or a composite score of 0  on day 31. The composite score was calculated from the maximum symptom scores for each symptom  after a lactose challenge test. Each symptom was rated on a 10-point Likert-type scale over 5 h after a lactose challenge. Each maximum symptom was then averaged into a composite score ranging from 0 to 10, where 0 indicated no symptoms and 10 indicated symptoms at their worst. A 4-point change was considered a meaningful improvement in symptoms of LI, based on psychometric analysis  and two rounds of cognitive interviews (N = 30 and 23). In the cognitive interviews, a 4-point change was meaningful for 84% of the subject responses and 53% of the subject responses in the first and second round of interviews, respectively. Symptom resolution was also considered meaningful. The 4-point threshold was further supported by empirical cumulative distribution functions using patient global severity anchor and was associated with sensitivity of 71%, specificity of 68%, positive predictive value of 63%, and negative predictive of 75%.Statistical analysis used two-tailed tests at the α = 0.05 level of significance. The proportions of responders on day 31 were analyzed by a stratified (by quartile of baseline LI symptom composite score) Cochran–Mantel–Haenszel (CMH) test comparing both doses combined versus placebo, higher dose versus placebo, and lower dose versus placebo. Prior to unblinding, the protocol was modified to specify that the primary endpoint was a comparison of the 2 active arms combined versus placebo, using a two-sided test at the α= 0.05 level of significance. Pooling the data provided greater statistical power. The modified intent to treat (mITT) analysis population was all randomized subjects who received at least 1 dose of drug.The sample size of 372 subjects was designed with the standard deviation of percentage abdominal pain reduction being 50%. It was hypothesized that the mean percentage reduction reported would be 50% for placebo-treated subjects and 70% for actively treated subjects. A simulation using Dunnett comparisons indicated that with 113 evaluable subjects in each arm, there would be 90% power to detect at least 1 of the active arms to be superior to placebo. The primary endpoint was based on a dichotomization of the distribution of the composite symptom score, which was thought to have at least as much power as percentage abdominal pain reduction.The SAS software version 9.4 was used.A number of secondary analyses were conducted. The proportion of participants in each group reporting no symptoms by the lactose intolerance composite score as well as the individual symptoms of abdominal pain, cramping, bloating, and gas movement after lactose challenge on day 31 was determined. The amount of milk consumed by participants on day 61 after RP-G28 or placebo was assessed. Global endpoints as defined in Bifidobacterium and Lactobacillus species was computed based on the Livak method. Quantitation cycle (Cq) values for each sample were normalized against the Cq value for the universal primers. Fold differences were calculated by 2 –ΔΔCt. Paired t-test and ANOVA with Tukey tests were conducted to assess statistically significant differences between groups.Paired-end fastq files were joined into a single multiplexed, single-end fastq using the software tool fastq-join. Demultiplexing and quality filtering were performed on the joined results. Quality analysis reports were produced using the FastQC software . BioinfoAll treated subjects were included in the safety analyses using summary statistics by treatment group of AEs, concomitant medications, vital signs, physical examinations, and clinical laboratory measurements. AEs were reported from initiation of treatment through 30 days of the post-treatment period. An AE was classified as a Severe Adverse Event if it interfered significantly with the patients’ usual functions.p = 0.04). In addition, patients reported higher symptom severity scores during the in-clinic lactose challenge compared to the other site’s subjects (p = 0.035). High milk consumption in addition to high symptom severity scores based on a blinded in-clinic lactose challenge is inconsistent with patients with LI. Further, the screen failure rate was also significantly lower at this center compared to other centers.A for-cause audit of one study site (out of 15 sites) was conducted due to significant data irregularities. The audit found significant deviations from the protocol and failure to comply with regulatory requirements for Good Clinical Practice (GCP), and identified protocol deviations including medical history and concomitant medications, inclusion/exclusion entry criteria, and administration of the Hydrogen Breath Test (HBT), lactose challenge, and patient diaries. Compared to the other sites, there were significant differences in multiple baseline symptom scores at this site. Patients reported two times more dairy intake prior to entry into the trial compared to subjects at other centers , and 344  completed the study. A high screen failure rate (>70%) was expected based on the rigorous inclusion and exclusion criteria required, including testing positive for LNP from a HBT and meeting LI symptom thresholds (63% of screen failures were due to HBT or LI symptoms scores not being met).p = 0.016). In total, 41% of subjects treated with lower-dose RP-G28 (p = 0.043) and 38% of subjects treated with higher-dose RP-G28 (p = 0.029) responded. (p = 0.062 (40% with treatment versus 31% with placebo).In the Efficacy Subset mITT group, significantly more patients in the pooled RP-G28 group responded as compared to patients in the placebo group—40% versus 26% (sponded.  In the mp = 0.004) and individual symptoms of abdominal pain (p = 0.014), abdominal cramping (p = 0.002), abdominal bloating (p = 0.015), and gas movement (p = 0.001) than placebo  and bloating (p = 0.028). Non-significant trends for improvement were seen for abdominal pain (p = 0.105) and gas movement (p = 0.060) (Patients treated with RP-G28 exhibited a consistent and significantly greater decrease in LI symptoms including cramping (= 0.060) B.p = 0.008, Efficacy Subset mITT) , patients treated with RP-G28 reported drinking significantly more milk, drinking an average of 1.5 cups/day versus 0.2 cups/day prior to treatment. The mean increase in milk intake of 1.3 cups/day (SD 1.479) in treated patients was significantly higher than that of the placebo group, which was 0.7 cups/day (SD 1.591) (et mITT) C.p = 0.01, Efficacy Subset mITT)). Patients treated with RP-G28 also consumed more dairy in general, ingesting 5.4 cups/day on day 61 versus only 1 cup prior to treatment, at baseline. In comparison, the placebo group ingested 4.5 cups/day on day 61 versus 1.7 cups/day at baseline. The mean increase in dairy intake of 4.3 cups/day trended toward significance compared to the placebo group .In addition, 59% of treatment patients consumed ≥1 cups/d of milk after being treated with RP-G28 in comparison to 42% with placebo . In addition, significantly more treated patients reported satisfaction with the ability of RP-G28 to prevent or treat their LI symptoms, with 66% of treatment patients reporting “very satisfied” or “extremely satisfied” as compared to 52% in the placebo group . Patients’ perception of adequate relief from LI symptoms and patients’ global impression of change were also improved with treatment   reported “no symptoms” or “mild symptoms”, respectively) as compared to 64% in the placebo group after treatment (et mITT) .p < 0.05). Of those, relative abundances of five Bifidobacterium taxa  were increased by both RP-G28 treatments. High-throughput qPCR quantitative data confirmed a significant (p < 0.05) increase in the abundance of the phylum Actinobacteria, the family Bifidobacteriaceae, and the genus Bifidobacterium on day 31 in the higher- and lower-dose treatment groups, but not the placebo group  of patients treated with RP-G28 had elevated Bifidobacteria levels, as compared to 52% (49/94) of patients in the placebo group (p < 0.001). Data using Firmicutes, Lactobacillaceae and Lactobacillus species-specific 16S rRNA gene probes showed a significant increase in the relative abundance of the family Lactobacillaceae on day 31 in the lower-dose treatment and a non-significant increase in the higher-dose group, but not in the placebo group.Of 543 bacterial species identified overall, 28 were differentially represented  with no differences between placebo and RP-G28. None of the serious adverse events (SAEs) were treatment related. No TEAEs resulted in hospitalization or death .Lactobacillus and Bifidobacterium [Though lactose intolerance is remarkably common, treatment options have not changed for decades ,17. The acterium . Increasacterium ,17, and acterium ,18.To assess the efficacy and meaningfulness of treatment, three broad groups of LI patient experiences were evaluated—a LI composite score to measure specific symptoms, milk and dairy intake to evaluate patients’ ability to increase dietary lactose with treatment, and global assessment measures to assess patients’ overall satisfaction with therapy. This three-pronged assessment provided a comprehensive evaluation of how treatment influenced symptoms of LI and the day to day lives of patients.A composite symptom score and a stringent threshold of meaningful change was established prior to unblinding the study in order to identify patients who reported a meaningful decrease in LI symptoms. Psychometric analyses indicated that the four symptom severity items were distinct yet related enough to support the creation of a composite score, and this composite score was shown to be reliable, valid, and responsive to change in LI symptoms over time. This tool provides a valuable resource for accurately assessing symptoms from the patient perspective in future LI clinical trials, and for identifying subjects who have experienced a meaningful treatment benefit in terms of patient-reported LI symptom severity. This study is one of the first to define and measure a meaningful treatment benefit for LI patients. The tool was developed adhering to good measurement principles following the FDA PRO requirements , and witp < 0.05).In the Efficacy Subset mITT population, the primary endpoint of response on day 31 was achieved in the pooled and individual RP-G28 groups. Individual symptoms were consistently reduced. The most rigorous endpoint for symptom relief is complete elimination. In the Efficacy Subset mITT population, RP-G28 administration resulted in complete symptom relief in a significantly greater proportion of patients than placebo, for the measures of abdominal pain, cramping, bloating, gas movement and overall symptoms (RP-G28-treated patients drank significantly more milk after treatment compared to the control group, drinking an average of 1.3 cups/day, as compared to an average of 0.7 cups/day more by the placebo group. Milk is the primary source of lactose in the diet, and thus milk consumption is an important indicator of the clinical benefits of supplementation with RP-G28. The ability to drink milk without LI symptoms supports optimal nutrient intake, contributing to the USDA’s recommended 2–3 cups of dairy per day . The susConsistent, statistically significant improvement in global assessments support the clinical meaningfulness of RP-G28. When consuming dairy foods for 30 days after treatment, 82% of patients reported “no or mild symptoms”, 66% reported being “very or extremely satisfied,” 83% reported adequate relief and 40% reported “very much or much improvement”.Overall, RP-G28 was safe and well tolerated. There were no differences in adverse events in those receiving RP-G28 or placebo. There were no serious adverse events in either group. Of note, there were no increases in GI side effects with RP-G28. This is important as RP-G28 is a GOS and, thus, part of the family of fermentable, oligo, di, monosaccharides, and polyols referred to as FODMAPs. FODMAPs have been shown to trigger GI symptoms in patients with irritable bowel syndrome. It is reassuring that at the doses administered, RP-G28 did not induce any significant GI side effects.Bifidobacteria, and Lactobacillaceae. Patient microbiomes adapted further with the reintroduction of lactose into patient diets during a 30 day period post-treatment, with an increase in lactose-fermenting Roseburia species [Bifidobacterium and Lactobacillus resulting from RP-G28 treatment, known to enhance lactose fermentation, is likely instrumental in better clinical outcomes. Further, stool from patients adapted to lactose produce less hydrogen, due to an absolute reduction in hydrogen production, rather than an alteration in hydrogen uptake by the microbiome [Gut microbiome changes and a reduction in net hydrogen gas production support the hypothesized mode of action . In tota species ,12. Elevcrobiome . AdditioOne of the 15 study sites was excluded from the Efficacy Subset mITT due to significant GCP violations. Nevertheless, this is the largest and most rigorously designed double-blinded randomized trial for the treatment of LI ever conducted. Another limitation is that the construct that identifies LI is subjective, depending on a single lactose dose and a subsequent time course. Real-world LI is likely intermittent, and depends on diet, dose, transit and other environmental and biological factors that are impossible to fully control within a clinical trial. However, in this study, the primary endpoint is part of a comprehensive assessment, including primary and secondary efficacy endpoints, global patient assessments, lactose consumption assessments, and correlation of treatment to changes in the microbiome—all of which are indicative of a beneficial effect of RP-G28 in improving symptoms of LI.Another limitation of the study is the duration, which does not allow determination of long-term durability. While significant improvements in diet quality and lactose tolerance were evident on days 31 and 61, we cannot determine whether retreatment will be necessary or effective.RP-G28 was safe and effective for reducing or eliminating symptoms of LI. Treatment with RP-G28 led to increased milk and dairy intake and improved quality of life. RP-G28 was safe and well tolerated at the doses administered. RP-G28 led to changes in the microbiome, which may be involved in the clinical benefits observed in patients with LI. These findings are relevant not only to lactose intolerance, but also usher in an era of using prebiotics to manipulate the microbiome to facilitate gut health.


However, there were no differences in CMJ ) and run-up jump ). Spiking speed was significantly reduced ), although accuracy was improved (38.3% ± 81.4%: p < 0.05) at T48. Thus, the initial, preseason RT bout compromised agility and spiking speed for several days post-exercise. Conversely, spiking accuracy improved, suggesting a speed–accuracy trade-off. Nonetheless, at least a 48-h recovery may be necessary after the initial RT bout for athletes returning from the off-season or injury.This study examined the acute effects of resistance training (RT) on volleyball-specific performance. Sixteen female volleyball players undertook their initial, pre-season RT bout. Countermovement jump (CMJ), delayed onset of muscle soreness (DOMS), and sport-specific performances  were measured before, 24 (T24), and 48 (T48) hours after RT. A significant increase in DOMS was observed at T24 and T48 (~207.6% ± 119.3%;  Volleyball is a team sport involving high-intensity, intermittent bouts of different activities, interspersed with longer recovery periods . These rStudies have reported high levels of EIMD with concomitant impairments in running economy ,16,17, rMore recently, a study by de Freitas et al.  examinedThis study was conducted as a repeated measures design across two weeks. The first week consisted of a familiarization session and a baseline assessment session. During the familiarization session, the athletes were acquainted with all protocols incorporated in the study, and a one-repetition maximum assessment was conducted for all the resistance exercises performed during the subsequent week. The baseline assessment session (TBase) consisted of indirect muscle damage markers, vertical jump, agility performance, and spike-specific performance protocols. During the second week, at least three days following TBase, a whole-body RT bout was undertaken, followed by assessment sessions equivalent to that of the baseline assessment session at 24 h (T24) and 48 h (T48) after the RT bout. To minimize potential influence of exercise on EIMD, no training was undertaken for up to 48 h following the RT bout.The participants were 16 female college volleyball players  who competed in national level competitions. All participants played volleyball in highly competitive events for 10.2 ± 2.7 years and had 5.5 ± 2.1 years of experience undertaking RT. At the time of the study, they were returning from a 3-week off-season. During the off-season, the participants continued undertaking volleyball-specific conditioning  1–2 times per week for three weeks, but either refrained from a structured RT program, or substantially reduced their RT volume by at least half, as typically instructed by volleyball coaches for off-season recovery. Once participants received the general background of the study, testing procedures, and potential risks involved, they provided informed consent. All experimental procedures were approved by the Experiment Ethics Committee of Chukyo University , and were conducted in accordance with the Declaration of Helsinki.The indirect muscle damage markers included delayed onset of muscle soreness (DOMS) of specific muscle groups , gluteal muscles (DOMSGts), triceps (DOMSTri), abdominals (DOMSAbs), and squat soreness (DOMSSqt)). The DOMS for each muscle group was assessed with the following technique: DOMSQuad—isometric contraction of the right quadricep muscles with the knee in an extended position; DOMSGts—isometrically contracting the gluteal muscles in a standing position; DOMSTri—performance of one push-up; DOMSAbs—performance of one sit-up; and DOMSSqt—overall lower body soreness during one bodyweight squat. The level of DOMS was assessed using a visual analogue scale from 1–10, with 1 defined as “no soreness” and 10 as “very, very sore” ,21. The The sport-specific performance measures included the countermovement jump (CMJ) test, run-up jump protocols (VJRUN) and a T-agility test protocol. The CMJ test was conducted with two maximal attempts, interspersed using a custom-built vertical jump apparatus . This vertical jump apparatus consisted of a wooden pole screwed to a wall, attached with pegs separated by 1 cm. Participants were provided with 30–60 s of rest in-between each CMJ attempt, and the best score was recorded for later analyses. To ensure standardization, the participants were instructed to maintain their heels in contact with the ground during the eccentric phases prior to take-off, and to come off the floor with their knees and ankles fully extended, sustaining a correct posture and body alignment throughout the movement. The VJRUN was completed by having participants run towards the vertical jump apparatus from a 3 m distance, leaving the floor with two feet  and tapping the pegs with the hand used for spiking the ball . Two attThe spike-specific performance test was undertaken on a standard volleyball court (9 m × 18 m) and the net height was set for female volleyball players (2.24 m) . As partThe participants commenced with a progressive warm-up by jogging at a self-selected pace for 2–3 min, followed by dynamic stretches of the upper and lower extremities . After the warm-up, the 1RM tests were conducted in the following order: power cleans, deadlifts, seated dumbbell shoulder press, and back squats, as in previously-described methods . SpecifiThe RT bout consisted of upper and lower body exercises, which were performed in order: power cleans (three sets of five repetitions at 80% of 1RM), deadlift (five sets of five repetitions at 80% of 1RM), dumbbell shoulder press (three sets of ten repetitions at 70% of 1RM), back squats (five sets of five repetitions at 80% of 1RM), and dumbbell lunges (four sets of ten repetitions on each leg at ~40% of body mass). Approximately two minutes of rest was provided in-between each set of each exercise. If participants perceived that they were able to undertake more of the assigned repetitions in a set, or were unable to complete the assigned repetitions in a set, the load was adjusted by 2–5% for the subsequent working sets.The continuous measures were reported as mean ± standard deviation, and the accuracy of the spiking protocol  was reported as frequencies via a cross-tabulated table. Data normality was checked using the Shapiro–Wilk test, and then analyzed using the Statistical Package of Social Sciences . A two-way (day × spike) repeated measures analysis of variance (ANOVA) was used to compare ball speed of the volleyball-specific performance protocol between testing days  and the five spiking attempts on each day. A one-way repeated measures ANOVA was then conducted for DOMS and SR and the sport-specific performance measures . For post-hoc analyses of the repeated measures ANOVA, Bonferonni’s pairwise comparison was employed. Effect size  with 95% confidence interval (CI) was also calculated to examine the magnitude of differences in the dependent measures for interaction and main effects, with 0.2 considered as a small ES, ≥0.5 as a moderate ES, and ≥0.8 as a large ES . To compp < 0.01; p > 0.05) with small ES  when compared to TBase with small ES , and between T24 and T48 (p > 0.05) with a small ES (0.07). For VJRUN, no significant differences were found at T24 ; p > 0.05; ES = 0.08 (95% CI: −0.77–0.62)) when compared to TBase, and between T24 and T48 ). However, time-to-completion for the T-agility test significantly increased at T48 when compared to TBase ; p > 0.05; ES = 0.10 (95% CI: −0.80–0.59)) and between T24 and T48 ). There was no interaction effect for the ball speed during the spike-specific performance protocol . However, a main effect was found between days  when compared to TBase ). For the accuracy of the spike-specific performance protocol, there was a significantly greater number of successful attempts at T48 (87.5%) when compared to TBase , although the participants exhibited a similar number of successful attempts between TBase and T24  and between T24 and T48 (p = 0.19).The CMJ exhibited no differences at T24 . However, no differences were found in vertical jump performance measures, whilst improvements were observed in spike accuracy. Accordingly, sufficient recovery should be considered when incorporating court-specific conditioning exercises that involve change-of-direction movements and spiking drills following the initial RT bout for elite, female volleyball players returning from the off-season.In the current study, the presence of DOMS is particularly interesting, given that the participants were resistance-trained with only three weeks of deconditioning as a result of the off-season. As far as we are aware, no studies have acutely monitored indirect muscle damage markers in elite, resistance-trained athletes returning from the off-season. Whilst it has been reported that individuals exposed to RT are protected from symptoms of EIMD for 6–9 months post-exercise due to the repeated bout effect , our finWhilst DOMS was elevated as a result of the RT bout, CMJ and VJRUN were unperturbed in the current study. These findings were unexpected, given that symptoms of DOMS have been shown to impair muscular contractility ,31. FurtAs opposed to the CMJ and VJRUN, agility performance was impaired following the bout of RT. This discrepancy between VJRUN and agility performance may be due to differences in the complexity of the task constraints. For example, our participants were familiar with agility tasks, as change of direction capabilities are essential in volleyball , similarn = 16), which may have provided sufficient statistical power to detect differences between time points. Despite equivocal findings with literature in elite volleyball players, our findings suggest that the initial RT bout compromises agility performance for up to 48 h post-exercise following several weeks of off-season-related deconditioning.Interestingly, de Freitas et al.  recentlyWith respect to the spiking-specific performance measures, the RT bout reduced ball speed whilst concomitantly improving accuracy. This decrement in speed at the cost of a heightened accuracy can be explained according to Fitt’s theory of the speed–accuracy trade-off  during tThe current study demonstrated that a bout of RT induced DOMS, impaired agility performance, and reduced ball speed during a spike-specific protocol for 48 h post-exercise in elite, female athletes returning from the off-season. Subsequently, athletes deconditioned from several weeks of RT  may require more than 48 h of recovery following their initial bout of moderate-to-high intensity (~75% of 1RM) RT. This is particularly important if the RT bout is implemented prior to an important volleyball match , or a conditioning session involving high-intensity multidirectional exercises. In addition, athletes in the off-season are recommended to undertake at least one resistance training session a week at similar intensity and volume to their competitive season, where possible, to mitigate high levels of EIMD from their initial RT session in the pre-season. Studies have also reported accelerated recovery following strenuous training sessions, including massage, warm-down ,45, and


Sleep disturbance and sleep disruption are associated with chronic, low grade inflammation and may underpin a range of chronic diseases in night shift workers. Through modulation of the intestinal microbiota, probiotic supplements may moderate the effects of sleep disruption on the immune system. The aim of this study was to examine 14 days of daily probiotic supplementation on the acute response of acute phase proteins and immune markers to sleep disruption associated with night shift work .10 colony forming units (CFU) Lactobacillus acidophilus DDS-1 or 1 × 1010 CFU Bifidobacterium animalis subsp. lactis UABla-12) or placebo (n= 29 per group). Participants undertook a 14-day supplementation period that coincided with a period of no night shifts followed by two consecutive night shifts. Blood samples were collected prior to the start of supplementation (V1), prior to commencing the first night shift (V2), after the first night shift (V3) and after the second night shift (V4). Serum was assessed for markers of stress (cortisol), acute phase response (C reactive protein (CRP), erythrocyte sedimentation rate, pentraxin), adhesion markers , and serum cytokines (interleukin (IL)-1ra, IL-1β, IL-6, tumor necrosis factor (TNF)-α, IL-10). Sleep quality was assessed with the Pittsburgh Sleep Quality Index (PSQI) and a Fitbit activity tracker.Individuals  performing routine night shift were randomly assigned to a probiotic group  was associated with significant changes in the placebo group in the concentration of serum cortisol (p = 0.01), pentraxin (p = 0.001), MAdCAM-1 (p = 0.001), and IL-1ra (p=0.03). In contrast, probiotic supplementation moderated changes in these serum markers from V1 to V2. No significant interaction effects (time by group) were observed for the serum markers prior to and after night shift work following probiotic supplementation due to the substantial changes in the serum markers that occurred during the normal sleep period from V1 to V2.Probiotics may moderate the effects of anticipatory stress on the immune system in the lead up to night shift. Night shift work is associated with a range of adverse health outcomes, including obesity and metabolic disease, cardiovascular disease and depression . Night s+ T-cells  that controls circadian rhythm. Systemic levels of key immune regulatory cytokines exhibit a circadian profile with peaks at mid-morning and late afternoon , reflectThe microbiota plays a key role in the ontogeny of the immune system and the regulation of inflammation throughout life. Recognition that direct interaction of commensal bacteria with epithelial cells modulates the mucosal cytokine milieu and that microbial metabolites induce regulatory T-cells –13 has lLactobacillus acidophilus DDS-1 and Bifidobacterium animalis subsp. lactis UABla-12, on indices of the immune system in individuals undertaking night shift work. L. acidophilus DDS‐1 and B. lactis UABla-12, alone or in combination, have previously been shown to reduce abdominal pain severity and symptomology in irritable bowel syndrome . Blood samples were collected at baseline (V1) (before probiotic intervention), before (V2) and after (V3) the first night of sleep disruption and again after the second night (V4) of sleep disruption Figure 1Individuals undertaking night shift work, including medical staff, transport, emergency services and security workers were contacted by email alerts with the invitation to participate in the research. Participants were required to be between 18 and 65 years of age and engaged in a rotating work roster that included night shift and day shift work. Individuals using immune-modulatory medications, sleep medications, antibiotics, who had taken probiotics in the month prior to the study or who had gastrointestinal or metabolic conditions, were pregnant or had a current bacterial or viral illness were excluded from participation.10 colony forming units; cfu) of either Lactobacillus acidophilus (DDS-1), Bifidobacterium animalis subsp. lactis (UABla-12) or a placebo, identical in appearance. Compliance was assessed with a daily checklist and the return of the remaining capsules for counting at the end of each participant’s study period.Participants were required to consume one capsule daily  and heparinized tubes  and PaxGene tubes . Plasma and serum were separated by centrifugation at 3500g for 10 min and stored frozen at −80°C until analysis.Blood samples were collected between 06:00 and 09:00 hours on the sample collection days. Blood samples were collected into K3EDTA tubes , serum separator , intestinal fatty acid binding protein , pentraxin , serum alpha amyloid , E-selectin ; interleukin(IL)1β, IL-6, IL-10, IL-1ra, tumor necrosis factor-alpha (TNF-α), and cortisol were determined using commercially available enzyme-linked immunosorbent assays (ELISA) and suspension array kits  according to the manufacturer’s instructions. Dilution factors were optimized for each assay to ensure an acceptable goodness of fit to the standard curve. For each analyte, samples were assessed in duplicate  and all samples from a single individual were assessed on a single assay plate. Inter-assay coefficients of variation ranged from 6.6% to 13.3%.Determination of C-reactive protein (CRP) concentrations was completed using a COBAS Integra 400 system and commercially available reagents, calibrators and controls (Roche Diagnostics). Concentrations of other soluble analytes, including lipopolysaccharide binding protein  were used for microbial identification. Sequence specific primers and probes were used as described previously  value was determined for duplicate samples and the change in CT values (ΔCT) between pre- and post-intervention visits determined. For the L. acidophilus and B. lactis reactions, the ΔCT was normalized against the ΔCTvalue obtainedfrom the universal bacterial reaction to standardized for sample input and converted to a fold change value.A fecal sample and 3-day food diary were collected from each participant prior to supplementation and at day 14. Participants were provided with a collection kit with instructions not to contaminate the sample with urine or water and to store the sample at room temperature until their study visit. Stool samples were frozen at −80°C upon receipt until processing. For probiotic bacteria identification, samples were brought to room temperature and total DNA was extracted from 2.0 g of fecal material using the QIAGEN DNA Stool Mini Kit  according to the manufacturer’s instructions. Following extraction, DNA concentrations were determined using a Nanodrop Spectrophotometer . Isolated fecal DNA was used for the detection of the bacterial species eviously . Reactiond probe  was alsoF ratios) were assessed using a Student’s paired t-test. Post-hoc analysis was undertaken using a Student’s t-test or appropriate non-parametric tests. Statistical significance was accepted at p=0.05.Statistical analyses were performed with IBM SPSS Statistics for Windows  and GraphPad Prism Version 7.00 for Windows, . Given that substantial inter-individual variability is reported in the literature for serum cytokines and other soluble immune markers  our sampvia Fitbit monitoring per day at V1  were included in the final analysis, as detailed in the CONSORT diagram Table 1.L. acidophilus DDS-1 in the DDS-1 group (median increase: 7.7-fold post-supplementation) was noted compared to the UABla-12 (median increase: 1.5-fold) and PLA (median increase: 1.8-fold) groups. Similarly, a difference in the recovery of Bifidobacterium lactis (UABla-12) in the UABla-12 group (median increase: 1700-fold post-supplementation) was noted compared to the DDS-1 group (median change: 80% decrease) and the PLA group (median increase: 1.3-fold). Large inter-individual variation in the recovery of both DDS-1 and UABla-12 was noted.Fecal samples were collected at V1 and V2 with changes in the recovery of the probiotic species assessed as a surrogate measure of compliance with supplementation. A difference in the recovery of Probiotic supplementation appears to have moderated changes in a number of serum analytes between V1 and V2. As shown in A significant time by group effect was observed for changes in the PSQI between pre-supplementation (V1) and end (V4) of supplementation  = 4.32; p = 0.018). Post-hoc analysis revealed that UAB-la12 was associated with a statistical trend for a 22% (p = 0.06) decrease in the PSQI Figure 3The aim of this study was to investigate the acute effects of probiotic supplementation on indices of stress, the acute phase response and inflammation during two nights of night shift. While no between group differences over the course of the study were observed in the serum analytes, there were a number of significant within-group changes observed between V1 and V2, with the largest changes observed in the placebo group. The period between V1 and V2 was when patients were supplementing in the lead up to night shift and adhering to a night-day sleep-wake cycle. The effects observed between V1 and V2 are consistent with an anticipatory stress effect prior to the start of night shift . AnticipGiven our study involved healthy individuals, the 10% to 70% changes in the concentration of a range of serum markers, in particular cortisol, pentraxin, MAdCAM-1, IL-1ra, and LBP, across all three groups during a typical night-day sleep-wake cycle (between V1 and V2) was unexpected. Further to this, the anticipation-induced changes in the serum markers was greater than that induced by night shift; an outcome that limits interpretation of other differences between the groups following night shift. The changes in serum markers in the lead up to night shift is consistent with understanding of anticipatory stress on sleep, hormones and immune indices , 32. AntBifidobacterium genus (over Lactobacillus), in part due to a more favorable environment of the large intestine  suggested as a potential mechanism  and the Gold Coast Hospital and Health Service Ethics Committee (HREC/17/QGC/271). The patients/participants provided their written informed consent to participate in this study.Study design and concepts: NW, AJC, AWC, GL, CM. Data analysis and interpretation: NW, AJC, AWC, PZ, RR, LH. All authors contributed to the article and approved the submitted version.This study was funded through a grant to Griffith University by UAS Laboratories.Authors GL and CM are employed by UAS Laboratories LLC.The authors declare that this study received funding from UAS Laboratories LLC. The funder had the following involvement with the study: Input into the study design and manuscript drafting. The funder was not involved in the collection of samples and data, sample and data analysis or the interpretation of data.https://www.frontiersin.org/articles/10.3389/fimmu.2020.599547/full#supplementary-materialThe Supplementary Material for this article can be found online at: Click here for additional data file.


Deregulation of miRNA-21 expression has been reported to be associated with vascular smooth muscle behavior and cytoskeletal stability. This study is aimed to investigate the density of serum miRNA-21 in patients with different phases of intracranial aneurysms (IAs) and explore its warning function for IA rupture.n = 40) for miRNA-21 validation. Potential gene targets of miRNA-21 were screened out from Gene Ontology (GO) database and literatures.A total of 16 in 200 IA patients were selected and categorized into 4 groups based on the phase of IA. Microarray study was carried out using serum miRNA and differentially expressed miRNAs were identified. Another 24 samples from a cohort of 360 patients were added and real-time polymerase chain reaction (RT-PCR) was performed on expanded sample size (Microarray study identified 77 miRNAs with significantly different expression levels between experimental groups and the control group. RT-PCR assays validated significant downregulation of miRNA-21 in experimental groups, among which miRNA-21 expression level of daughter aneurysm group decreased the most. Bioinformatic analyses revealed that several target genes related with miRNA-21 may be involved in IA formation and rupture.This study suggested that miRNA-21 had a protective effect for intracranial vascular wall against remodeling and warning function for intracranial aneurysm rupture. Significant suppression of serum miRNA-21 in IA patients may provide diagnostic clues for aneurysm rupture and guide clinical intervention. Pathological changes of intracranial aneurysm (IA) include vascular smooth muscle cells (VSMC) apoptosis, matrix degradation, inflammation, and oxidation in the artery wall . HoweverMicroRNA (miRNA) is a highly conserved small non-coding single-stranded RNA (20–26 nucleotides in length). It regulates genetic transcription and translation via inhibition or messenger RNA degradation . More thDeregulation of miRNA-21 expression was reported to be associated with vascular smooth muscle behavior and cytoskeletal stability through regulating tropomyosin- 1. ResearcThe study was approved by the Ethics Committee of our institution and performed in accordance with the principles of the Declaration of Helsinki. All the patients signed written informed consent.In the first step, we selected 16 from 200 IA patients at our department from January to June 2015. All the patients were screening out based on strict criteria . As was illustrated in Table n = 16, 40.0%), posterior communicating artery , anterior communicating artery , and posterior circulation . The size of the aneurysm ranges from 1.0 to 30.0 mm.In order to validate the expression difference of miRNA-21, we added the sample size with the other 24 samples selected under the same screening criteria from 360 patients from July to December 2015 . Qualities of miRNAs were assessed using NanoDrop 2000 spectrophotometer. One hundred nanograms of miRNA was taken and added to 2 μm with nuclease-free water for further analysis.Blood samples were collected in the morning, no drug interference and intravenous transfusion before operation. Blood samples (2 ml) of the peripheral vein were collected using EDTA tubes. Blood samples were centrifuged at 1200 g for 15 min immediately after collection. Supernatants were transferred into micro-centrifuge tubes. Second centrifugation was performed at 12,000×miRNAs were labeled with miRNA Complete Labeling and Hybridization Kit  and hybridized at Human miRNA Microarray Kit , which contained 60,000 probes for 144 human viral and 1205 human miRNAs. Microarray Scanner  was used to detect hybridization signals. Feature Extraction Software  was used for image analysis. Principal component analysis (PCA) and percentile normalization were performed using Gene spring 12.0. Cluster 3.0 was used for Cluster analysis and differentially expressed miRNAs were screened out.miRNA-21 was validated by RT-PCR and the small non-coding miRNA-16 was internal control. Primer sequence of miRNA-16 and miRNA-21 were 5′-TAGCAGCACGTAAATATTGGCG-3′ and 5′-TAGCTTATCAGACTGATGTTGA-3′. The reverse transcription was performed for miRNA-21 and miRNA-16 using the miScript II Reverse Transcription Kit . The reverse transcription products were used for the TaqMan real-time PCR reaction with Super Real PreMix . RT-PCR was performed using the Applied Biosystems 9700 RT-PCR system. The PCR conditions were 95 °C for 15 min, and then 10 s at 95 °C, and 30 s at 60 °C for 40 cycles.Gene Ontology (GO) database was used to predict genes related to miRNA-21. Network was generated to reveal high-level, summarized views of predicted target genes from the GO database and literatures.P < 0.05 is regarded as statistical significance.SPSS 18.0 is used for data analysis. The expression difference of miRNAs between groups is analyzed by one-way ANOVA and Dunnett’s multiple comparison test. p < 0.05) as revealed in Table P value of miRNA-21 is 0.027 and fold change (FC) of group B, C, and D are 1.51, − 2.31, and 1.25, respectively.A total of 77 miRNAs are statistically different , and group A and group D (p = 0.013). Histogram demonstrates that the variation tendency of miRNA-21 expression levels among different groups  is found.Relative expression levels of miRNA-21 of 40 samples are listed in Table miRNA-21 target gene analysis was performed using target prediction GO database. A total of 26 genes are closely related with miRNA-21 Fig. . miRNA-2miRNA-21 is one of the most commonly and markedly regulation factor in several cardiovascular disorders and cancers . ExpressDaughter aneurysm is recognized as a high-risk factor of IA rupture , which iCorrelation analysis shows no statistical significance between miRNA-21 expression level and aneurysm size. This agrees with clinical experiments that the rupture risk of aneurysm does not increase with the increase of size. Contradictorily, researchers proved that aneurysms of relatively smaller diameter had a higher risk of rupture than larger aneurysms , 16.It is worth noting that the difference between groups A and C is greater, while the difference between A, and B and D is relatively smaller. This may probably imply that a significant low expression level of miRNA-21 has a warning function for aneurysm rupture. Clustering analysis revealed that compared with group A, the expression of miRNA-21 is upregulated in group B and D (red), while downregulated in group C (green).The whole progress of aneurysm is a dynamic process. Control group stands for normal expression level of miRNA-21; that of group B is downregulated to a relatively low extent. Then, with the progress of aneurysm development, expression level of miRNA-21 in group C becomes even lower. After the rupture of an aneurysm, the expression of miRNA-21 rises up. Since group C is a relatively high-rupture risk group and miRNA-21 expression of this group is extremely low, the hypothesis could be developed that miRNA-21 could have a protective effect for vascular wall and warning function for aneurysm rupture. When the body received the impact of internal or external stimulus, miRNA-21 expression level becomes downregulated. As a result, its protection for vascular wall becomes weaker and aneurysm forms as a consequence of vascular remolding. If the stimulus lasts long enough, miRNA-21 expression will continue to fall, and daughter aneurysm formation and aneurysm rupture risk will increase. Expression of miRNA-21 rising up again after rupture may be because of some protective feedback mechanism activation in the body or removal of stimulus. The biggest suppression of miRNA-21 in group C deserves our attention because this may provide diagnostic and therapeutic clues for aneurysm rupture.During the whole process of this rigorous study, the result of the first part (microarray) is not totally consistent with that of the second part (RT-PCR). Compared with the control group, microRNA-21 expression of groups B and D is upregulated in microarray study, while that of RT-PCR is downregulated. This may result in selection bias or small sample size. Since quantitative method (PCR) is more accurate than qualitative method (microarray). And sample size is bigger in PCR. So the authors are more inclined to the result of RT-PCR.In all, 26 target genes that are closely related with miRNA-21 were screened out using the GO database. Vascular endothelial growth factor C (VEGFC) is directly related with the activity of vascular wall from GO analysis. In addition, miRNA-21 may regulate vascular smooth muscle activity via other targets such as phosphatase and tensin homolog protein (PTEN), Bcl-2, and PDCD-4  are also believed to be key pathophysiological processes leading to aneurysm formation and rupture . The proThe study had some limitations. There is some bias because the sample size is small. For example, the result of microarray study is not coinciding exactly with that of RT-PCR. The classification of the high-rupture risk group is lacking specific criteria. In addition, there are some limitations using serum miRNA as biomarkers. Moreover, the result and hypothesis of this study were not validated with protein analysis. Much effort is needed before the protective effect and warning function of miRNA-21 could be used in diagnostic and therapeutic applications in IA rupture.This study suggested that miRNA-21 may have had a protective effect for intracranial vascular wall against remodeling and warning function for intracranial aneurysm rupture. Significant suppression of serum miRNA-21 in IA patients may provide diagnostic clues for aneurysm rupture and guide clinical intervention.